Enough people have asked for this method to make it worth posting.

Andy Shand

I repeat

Differentiation is critical and it takes practice but it's worth it.  Sorry
folks I've lost the reference.  If Ronnie Houston is paying attention I'm
sure he'll know.


Technical tips

1. The sections may be "held" in 1% calcium chloride for up to 10 minutes
before being stained.

Stock solutions

Acid pre-treatment      Triz buffer pH 7.8
Calcium chloride (dihydrate) -     1.30g   Trizma base           6.05g
Potassium acetate  -     2.45g   Calcium chloride (dihydrate)-1.3g
Distilled water   - 500mls   Distilled water  - 500mls
Adjust to pH 4.5 with concentrated acetic acid.  Adjust to pH 7.8 with 5M
HCl.

1% Calcium chloride  0.18M Calcium chloride
    Calcium chloride -     2.646g
Sigma 221 buffer  Distilled water  - 100mls

Incubation medium
Distilled water   - 15mls
Potassium chloride  - 0.0925g
ATP (disodium salt)  - 0.038g
Calcium chloride (0.18M) - 2.5mls
Sigma 221 buffer  - 1.675mls
Adjust to pH 9.4 with 1M or 5M HCl.

METHOD

1. Treat air dried frozen sections with acid pretreatment solution (6 mins.)
2. Transfer slides through three rinses in Triz buffer, 2 mins. each.
3. Place slides in incubation medium pH 9.4 for 25 mins. at room temp.
4. Rinse in three changes of 1% calcium chloride 5mins.
5. Drain and stain slides in 0.1% toluidine blue for 90 seconds.
6. Rinse  very quickly (no more than seconds) in distilled water to remove
excess stain.
7. Dehydrate rapidly (5 dips each of 95% alcohol and two changes of 100%
alcohol).
8. Clear in two changes of xylene and mount in DPX.

Results

Type I flibres  - Turquoise   Type IIA fibres - Light red
Type IIB fibres - Violet  Type IIC fibres - Blue